Class ii hmg-coa reductase inhibitors and methods of use

ABSTRACT

Disclosed are compositions and methods for treating bacterial infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication Nos. 61/637,091 filed Apr. 23, 2012 and 61/637,697 filedApr. 24, 2012, each of which is incorporated by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

INTRODUCTION

Antibiotic resistant pathogenic bacteria such as vancomycin resistantEnterococcus (VRE), methicillin resistant Staphylococcus aureus (MRSA),and penicillin resistant Streptococcus pneumoniae (PRSP) are the mostcommon causes of hospital-acquired (nosocomial) infections in the UnitedStates and Europe. Nosocomial infections cause tens of thousands ofdeaths annually and result in billions of dollars in additional healthcare costs in the U.S. alone. Antibiotics currently used to treat theseinfections frequently cause nausea, vomiting, and diarrhea. Becausethese antibiotics are not specific for the pathogenic bacteria and killnormal flora, i.e., commensal bacteria, treatment of infections oftencauses oral, intestinal, or genital infections due to fungal overgrowth.Furthermore, antibiotics used to treat antibiotic resistant bacteria,such as vancomycin, have adverse side effects, including nephrotoxicity.

There is a need in the art for new antibiotics effective in treatingpathogenic microbial infections. The compositions and methods describedherein address that need.

SUMMARY

The present invention relates generally to compositions and methods fortreating infections by pathogenic bacteria.

In certain embodiments, the invention includes compounds having one ofthe following two structures:

wherein:R¹ is a hydroxyl, alkoxy, or substituted alkoxy;R² is H, alkyl, or substituted alkyl;R⁴ and R⁶ are independently selected from alkyl substituents of the type—(CH₂)_(m)—R^(C), wherein m=0-14, optionally having polar substituentssuch as OH, NR^(A)R^(B), OPO₃H₂, OSO₃H, PO₃H₂, SO₂H, and CO₂, or acyclohexyl or benzyl substituent, optionally having unsaturation and/orbranching in the alkyl chain;R⁵=OH, NR^(A)R^(B), or a halogen;R^(A) and R^(B) are independently selected from —(CH₂)_(n)H,—(CH₂)_(n)OH, —(CH₂)_(n) CH(CH₃)₂, —(CH₂)—C(CH₃)₃,—(CH₂)_(n)-cyclohexyl, and —(CH₂)_(n)-phenyl, wherein n=0-3,R^(C)=H, OH, NR^(A)R^(B), OPO₃H₂, OSO₂H, PO₃H₂, SO₂H, CO₂, CH(CH₃)₂,C(CH₃)₃, cyclohexyl, or phenyl;X, Y, and Z are independently selected from N, CH, and CR⁵, such thatthe core ring structure is selected from benzene, pyridine, pyrazine,pyridazine, and pyrimidine;A, B, D, and E are independently selected from N, CH, and CR⁴, such thatthe core ring structure is selected from benzene, pyridine, pyrazine,pyridazine, and pyrimidine, orQ=S, O, or NR^(A) and U, V, and W are independently selected from N andCR⁵, such that the core ring structure is selected from pyrrole,imidazole, pyrazole, furan, oxazole, isooxazole, thiophene, thiazole,and isothiazole.

In certain embodiments, the compound is provided as a pharmaceuticallyacceptable salt.

In certain embodiments, the compounds are provided as a pharmaceuticalcomposition comprising one or more of the compounds and apharmaceutically acceptable carrier.

In certain embodiments, the compounds are provided as a biocide havingactivity against biofilms and certain biofilm forming bacteria.

Advantageously, the compounds and pharmaceutical compositions haveantibacterial activity against Gram-positive pathogens, while lackingantibacterial activity against Gram-negative commensal bacteria.

In certain embodiments, methods for treating a bacterial infection in asubject are provided. The methods involve administering a pharmaceuticalcomposition of the invention in an amount effective to inhibit growth ofor kill the bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows binding of substrates in the active site of HMGR.

FIG. 2 shows binding of inhibitor N-bsha in the active site of HMGR.

FIG. 3 shows the structures of compounds synthesized and tested forinhibitory activity.

FIG. 4 is a representative time-kill curve of compound 2f against MRSAcultured in Mueller Hinton broth (MHB).

FIG. 5 presents the results of cytotoxicity assays showing the percentmean absorbance at 490 nm after incubating J774A.1 cells with variouscompounds.

DETAILED DESCRIPTION

The compositions described herein were designed to specifically inhibitClass II 3-hydroxy-3-methylglutaryl-coenzyme A reductase (II-HMGR),which catalyzes the conversion of HMG to mevalonate and is arate-limiting enzyme in the mevalonate pathway. Certain Gram-positivecocci, including S. aureus, E. faecalis, and S. pneumoniae, relyexclusively on the mevalonate pathway for the production of isopentenylpyrophosphate (IPP), a precursor required for cell wall synthesis.Knocking out the HMGR gene in Gram-positive bacteria causes the bacteriato become weak and to lose virulence, and to depend on an externalsource of mevalonate. In contrast, most Gram-negative bacteria,including most commensal bacteria, depend on an alternate pathway forIPP synthesis and lack II-HMGR. Inhibition of II-HMGR is advantageousbecause it allows specific targeting of Gram-positive pathogens overGram-negative commensal bacteria.

The isoprenoid pathway takes three molecules of acetyl-CoA to createisopentenyl-diphosphate, an important building block for moleculesinvolved in cell wall synthesis, energy metabolism and lipid transport.The pathway utilizing HMG-CoA Reductase is present in all eukaryoticcells and is the target of the cholesterol lowering statin medications.Most bacteria utilize an alternative pathway to makeisopentenyl-diphosphate that does not use the HMG-CoA reductase enzyme.However, certain Gram-positive bacteria, notably the multidrug resistantStaphylococcus aureus and Enterococcus faecalis have the eukaryoticpathway with an HMG-CoA Reductase, though the active site issubstantially different than the eukaryotic enzyme.

The mechanism of the reductase is to bind HMG-CoA and NADH to thesurface of the molecule, bringing the nicotinamide ring of NADH and theC1 carbon close together. This binding causes changes in the activesite, facilitating the reduction of the HMG-CoA by the NADH. The reducedNAD is replaced by a second NADH. A second reduction creates the finalproducts, free CoA and mevalonate

All eukaryotes have a mevalonate pathway for the synthesis of IPP thatemploys Class I HMGR (I-HMGR). Inhibition of I-HMGR by statins is usedto reduce cholesterol biosynthesis in humans. The active site of II-HMGRis substantially different than that of the eukaryotic enzyme. Becauseof these differences, the affinity of statins for I-HMGR is six ordersof magnitude higher for I-HMGR than for II-HMGR. Exploitation ofdifferences between the active sites of I-HMGRs and II-HMGRs allows forthe design of inhibitors specific for II-HMGR.

As described in the examples below, it was discovered that5-(N-(4-butylphenyl)sulfamoyl)-2-hydroxybenzoic acid (N-bsha orcompound 1) inhibits HMGR-II. X-ray crystallographic studies reveal thatN-bsha binds in the active site of class II HMGR and is competitiveinhibitor of II-HMGR. Analogues of N-bsha were synthesized an evaluatedfor the ability to inhibit II-HMGR catalyzed conversion of HMG-CoA tomevalonate. Those analogues exhibiting inhibition of II-HMGR were testedfor the ability to inhibit antibiotic resistant bacteria.

The active site of the enzyme is a groove on the surface of themolecule. As part of this binding, the active site becomes covered witha 50 residue ‘flap’ domain that serves to protect the intermediateproducts from hydrolysis (FIG. 1). The binding of the inhibitors of thepresent invention also closes the flap domain.

The inhibitor first identified (N-bsha) binds in the pocket thatordinarily holds the HMG portion of the HMG-CoA reductase ligand (FIG.2). The binding includes direct and water mediated hydrogen bonds aroundthe benzoic acid portion of the inhibitor, and hydrophobic interactionsaround the benzene ring and alkane tail.

In certain embodiments, compounds according to the present inventionhave one of the two following structures:

wherein:R¹=is a hydroxyl, alkoxy, or substituted alkoxy;R²=is H, alkyl, or substituted alkyl;R⁴ and R⁶ are independently selected from alkyl substituents of the type—(CH₂)_(m)—R^(C), wherein m=0-14, optionally having polar substituentssuch as OH, NR^(A)R^(B), OPO₃H₂, OSO₃H, PO₃H₂, SO₂H, and CO₂, or acyclohexyl or benzyl substituent, optionally having unsaturation and/orbranching in the alkyl chain;R⁵=OH, NR^(A)R^(B), or a halogen;R^(A) and R^(B) are independently selected from —(CH₂)_(n)H,—(CH₂)_(n)OH, —(CH₂)_(n) CH(CH₃)₂, —(CH₂)—C(CH₃)₃,—(CH₂)_(n)-cyclohexyl, and —(CH₂)_(n)-phenyl, wherein n=0-3,R^(C)=H, OH, NR^(A)R^(B), OPO₃H₂, OSO₂H, PO₃H₂, SO₂H, CO₂, CH(CH₃)₂,C(CH₃)₃, cyclohexyl, or phenyl;X, Y, and Z are independently selected from N, CH, and CR⁵, such thatthe core ring structure is selected from benzene, pyridine, pyrazine,pyridazine, and pyrimidine;A, B, D, and E are independently selected from N, CH, and CR⁴, such thatthe core ring structure is selected from benzene, pyridine, pyrazine,pyridazine, and pyrimidine, orQ=S, O, or NR^(A) and U, V, and W are independently selected from N andCR⁵, such that the core ring structure is selected from pyrrole,imidazole, pyrazole, furan, oxazole, isooxazole, thiophene, thiazole,and isothiazole.

In certain embodiments, R¹ is OH. In certain embodiments, R¹ is—O—(CO)NH₂, or —OCH₂(CO)NH₂.

In certain embodiments, R² is H.

In certain embodiments, the compound includes A, B, D, and E in a ringstructure selected from the group consisting of benzene, pyridine,pyrazine, pyridazine, and pyrimidine. In certain embodiments, thecompound includes A, B, D, and E in a benzene ring structure.

Extending the carbon tail (R⁴ or R⁶) or adding hydrophobic (phenyl orhexane) groups to the carbon tail of the inhibitors of the presentinvention has been found to lead to greater inhibition of II-HMGR.

In certain embodiments, the compound includes Q, U, V, and W in a ringstructure selected from pyrrole, imidazole, pyrazole, furan, oxazole,isooxazole, thiophene, thiazole, and isothiazole.

In certain embodiments, the compound includes A, B, D, and E in a ringstructure selected from the group consisting of benzene, pyridine,pyrazine, pyridazine, and pyrimidine, and R⁴ is an alkyl substituent ofthe type —(CH₂)_(m)H, wherein m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, or 14. In some embodiments, the alkyl substituent is a branchedalkyl chain with from 4 to 14 carbon atoms. In some embodiments, thealkyl substituent is at least partially unsaturated, i.e., comprises oneor more alkenyl and/or alkynyl groups.

In certain embodiments, the compound includes Q, U, V, and W in a ringstructure selected from pyrrole, imidazole, pyrazole, furan, oxazole,isooxazole, thiophene, thiazole, and isothiazole and R⁶ is an alkylsubstituent of the type —(CH₂)_(m)H, wherein m is 0, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, the alkylsubstituent is a branched alkyl chain with from 4 to 14 carbon atoms. Insome embodiments, the alkyl substituent is at least partiallyunsaturated, i.e., comprises one or more alkenyl and/or alkynyl groups.

In certain embodiments, R⁴ or R⁶ are C₆H₁₃, C₇H₁₅, C₁₀H₂₁, C₁₂H₂₅,(CH₂)₃CH(CH₃)₂, (CH₂)₄CH(CH₃)₂, or (CH₂)c-C₆H₁₁.

In certain embodiments, II-HMGR inhibitors kill or inhibit the growth ofGram-positive pathogens, e.g., MRSA and/or VRE, without affecting thegrowth of commensal Gram-negative bacteria, e.g., Escherichia coli.

In certain embodiments, the II-HMGR inhibitors selectively inhibitII-HMGR over I-HMGR, with at least a 100-fold greater inhibitoryactivity against II-HMGR vs. I-HMGR.

The inhibitors of the present invention may be formulated as apharmaceutical composition suitable for administration by any suitablemode of administration, including, for example orally (e.g., enterallyor sublingually), intravenously, intramuscularly, subcutaneously,transdermally, vaginally, rectally, intranasally, and the like.

In certain embodiments, inhibitors of the invention are prepared,purified, or formulated as a corresponding salt of the active compoundor prodrug, for example, a pharmaceutically-acceptable salt. Examples ofpharmaceutically acceptable salts are discussed in Berge, et al., J.Pharm. Sci., 66, 1-19 (1977). Unless otherwise specified, a reference toa particular compound also includes salt forms thereof. The term“pharmaceutically acceptable salt” refers to those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts of thecompounds of this invention include those derived from suitableinorganic and organic acids and bases. Examples of pharmaceuticallyacceptable, nontoxic acid addition salts are salts of an amino groupformed with inorganic acids such as hydrochloric acid, hydrobromic acid,phosphoric acid, sulfuric acid and perchloric acid or with organic acidssuch as acetic acid, oxalic acid, maleic acid, tartaric acid, citricacid, succinic acid or malonic acid or by using other methods used inthe art such as ion exchange. Other pharmaceutically acceptable saltsinclude adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄ alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium, quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and arylsulfonate.

In certain embodiments, compositions of the present invention comprise aII-HMGR inhibitor and a pharmaceutically acceptable excipient, which, asused herein, includes any and all solvents, diluents, or other liquidvehicle, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W.Martin (Mack Publishing Co., Easton, Pa., 1980) discloses variouscarriers used in formulating pharmaceutically acceptable compositionsand known techniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, e.g., having an undesirable biological effect or otherwiseinteracting in a deleterious manner with any other component(s) of thepharmaceutically acceptable composition, its use is contemplated to bewithin the scope of this invention. Some examples of materials which canserve as pharmaceutically acceptable carriers include, but are notlimited to, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, or potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates,waxes, polyethylene-polyoxypropylene-block polymers, wool fat, sugarssuch as lactose, glucose and sucrose; starches such as corn starch andpotato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols; such apropylene glycol or polyethylene glycol; esters such as ethyl oleate andethyl laurate; agar; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol, and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, shaping and/or packaging the product into a desired single-or multi-dose unit.

A pharmaceutical composition of the invention may be prepared, packaged,and/or sold in bulk, as a single unit dose, and/or as a plurality ofsingle unit doses. As used herein, a “unit dose” is discrete amount ofthe pharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject and/or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

In certain embodiments, the compounds of the invention may be formulatedfor use as a bacteriocidal composition for use on surfaces that tend toserve as a support for biofilms, e.g., catheters. The bacteriocidalcompositions include the compound and, for example, a suitable carrier.The bacteriocidal compositions may be provided in a form comprising theII-HMGR inhibitor in a concentration effective to kill or inhibit thegrowth of bacteria, or conveniently may be supplied in a concentratedform to be diluted prior to use.

The following non-limiting examples are intended to be purelyillustrative.

EXAMPLES Screening of Compounds Inhibition of II-HMGR

A library of 300,000 compounds was screened against E. faecalis II-HMGRto identify II-HMGR inhibitors. Compounds were screened for the abilityto inhibit E. faecalis II-HMGR from the mevalonate to HMG-CoA directionby measuring changes in NADPH using absorbance at 340 nm. Among thecompounds screened, 5-(N-(4-butylphenyl)sulfamoyl)-2-hydroxybenzoic acid1 (N-bsha) was found to be the best inhibitor of II-HMGR, with an IC₅₀of 5 μM.

Design of the Modified HMGR Inhibitors

The X-ray crystal structure of E. faecalis HMGR complexed with inhibitor1 shows that the hydrophobic para-butyl group occupies a hydrophobicpocket in the active site that normally accommodates the pantothenicacid portion of HMG CoA. Initial investigation of an analogue of 1lacking the butyl “tail” indicated that an aliphatic moiety may beimportant for inhibition of HMGR. A series of analogues of 1 wassynthesized (2a-m) in which the aliphatic tail was varied, as show inFIG. 3. In analogues 2a-h, the length of the linear alkyl chain wasvaried from 2 to 14 carbon atoms; in 2i-k, the aliphatic chain hasterminal branching, and in 2l and 2m cyclohexyl and phenyl rings,respectively, terminate the aliphatic tail. It was envisaged thatincreased hydrophobic character in the tail may increase affinity forthe HMGR active site, provided that the tail can be accommodated withinthe active site.

Examination of the HMGR co-crystal structure with 1 also revealed thepresence of a water-mediated hydrogen bond between the phenolic hydroxylof 1 and the Asn213 residue of HMGR, which is known to form hydrogenbonds to the NADPH cofactor in the co-crystal structure of HMGR with asubstrate analogue. To displace the bound water molecule, analogue 3 wasdesigned in which the phenolic hydroxyl is alkylated with an acetamidesubstituent with the intention of engaging Asn213 in a cooperativehydrogen bond between the two primary amides.

Synthesis of the Modified Inhibitors

All starting materials were purchased from Sigma Aldrich (St. Louis,Mo., USA), TCI America (Portland, Oreg., USA) or VWR (VWR Direct,Arlington Heights, Ill., USA). All solvents were dried by passagethrough an activated column composed of activated alumina and supportedcopper redox catalyst reactant, prior use. To analyze compounds purity,reverse phase HPLC (RP-HPLC) was performed using a Gilson instrument andVydac C-8 column, with HPLC grade acetonitrile and deionized water and0.1% trifluoroacetic acid. Flash chromatography was performed using230-400 mesh silica gel or prep-HPLC was used to purify compounds.Proton NMR spectra were obtained on a Varian Inova-300 or a BrukerARX-400 spectrometer at 300 MHz and 400 MHz, respectively.

The target molecules were synthesized using well-established chemistry(Scheme 1). Commercially available 1-iodo-4-nitrobenzene (4) was coupledwith terminal alkynes using Sonogashira coupling, forming phenylalkynes6a-m in high yields. Simultaneous reduction of alkyne and nitro groupsby catalytic hydrogenation over platinum oxide afforded substitutedanilines 7a-m. The only exception was 4-propylaniline (7b), which wascommercially available. Each aniline was reacted with methyl5-(chlorosulfonyl)-2-hydroxybenzoate (8), synthesized according to aliterature protocol, under Schotten-Baumann conditions to affordsulfonamides which were saponified to afford the target molecules(2a-m). As a control, the lead compound 1 was synthesized fromcommercially available p-butylaniline using this same protocol.

The synthesis of the ether 3, is shown in Scheme 2. The sulfonamide 9,previously made as an intermediate during our synthesis of lead compound1, was alkylated on the phenolic hydroxyl with iodoacetamide under basicconditions to afford the ether 10. Saponification of the methyl ester of10 afforded the desired target 3.

Methyl 5-(chlorosulfonyl)-2-hydroxybenzoate (6)

Salicylic acid (3.00 g, 21.7 mmol) was dissolved in MeOH (50 mL) andH₂SO₄ (6 mL) was added dropwise to the stirring solution. The resultantsolution was heated to reflux for 18 h and, after cooling to roomtemperature, the solvent was removed under reduced pressure. The residuewas diluted with H₂O (10 mL) and extracted with CH₂Cl₂ (3×10 mL). Thecombined organic layers were washed sequentially with saturated aqueousNaHCO₃ (10 mL), brine (10 mL) and water (10 mL), dried over Na₂SO₄, andconcentrated under reduced pressure. The crude methyl salicylate wasobtained in 85% as a colorless oil (2.80 g) and used without furtherpurification. ¹H NMR (300 MHz, CDCl₃) δ 10.7 (s, 1H), 7.84 (d, J=7.5 Hz,1H), 7.47 (t, J=8.1 Hz, 1H), 6.99 (d, J=8.1 Hz, 1H), 6.90 (t, J=8.1 Hz,1H) ppm.

To a solution of SOCl₂ (1.34 mL, 18.4 mmol) and CISO₃H acid (4.92 mL,73.6 mmol) cooled to −10° C. was added slowly the crude methylsalicylate. The reaction was stirred at room temperature for 18 h, afterwhich the brown reaction was poured slowly over ice (10 mL) that wasplaced in an ice bath and continually stirred with a glass rod until theresultant precipitate turned colorless. The precipitate was filteredunder vacuum and washed with cold water. The sulfonyl chloride 6 wasobtained as a colorless solid (4.18 g, 91% yield). ¹H NMR (300 MHz,CDCl₃) δ 11.55 (s, 1H), 8.56 (d, J=2.1 Hz, 1H), 8.10 (dd, 2.4, 9.0 Hz,1H), 7.19 (d, J=9.0 Hz, 1H), 4.04 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃)δ 168.9, 166.4, 134.9, 133.5, 130.5, 119.3, 112.5, 53.1 ppm.

General Procedure for Sonogashira Reaction

In a round bottom flask under nitrogen, 1-iodo-4-nitrobenzene (3, 300mg, 1.20 mmol), freshly recrystallized CuI (28.8 mg, 0.120 mmol) andPd(PPh₃)₂Cl₂ (42.1 mg, 0.06 mmol) were dissolved in THF (5.0 mL). Theterminal alkyne 2 (1.4 mmol) was added dropwise to the solution, whichgradually turned bright yellow. To the resultant solution NEt₃ (0.68 mL,4.84 mmol, 4.0 eq.) was added slowly, during which time the reactionbecame dark brown. After 2 h, the reaction was diluted with water (5 mL)and extracted with CH₂Cl₂ (3×5 mL). The combined organic layers werewashed with brine (5 mL) and water (5 mL), dried over Na₂SO₄ andconcentrated under reduced pressure. The product arylalkynes 4 werepurified by flash chromatography using 100% hexanes.

1-Ethynyl-4-nitrobenzene (4a). TMS-protected 4a was obtained as acolorless, crystalline solid (279 mg, 99%); ¹H NMR (300 MHz, CDCl₃) δ8.15 (d, J=8.7 Hz, 2H), 7.58 (d, J=9.0 Hz, 2H), 0.25 (s, 9H) ppm. ¹³CNMR (75 MHz, CDCl₃) δ 146.9, 132.5, 129.8, 123.3, 102.5, 100.4, 85.7,−0.43 ppm. Deprotection was done by dissolving 4a (279 mg, 1.30 mmol) in3:1 THF-MeOH (4 mL) and adding potassium carbonate (528 mg, 3.82 mmol)slowly. The reaction was stirred for 2 h, after which the mixture wasdiluted with Et₂O, washed with saturated NH₄Cl and H₂O, dried with MgSO₄and concentrated under reduced pressure. The resultant terminal alkyne4a (129 mg, 68%) was then used with no further purification. ¹H-NMR (300MHz, CDCl₃) δ 8.19 (d, J=8.4 Hz, 2H), 7.64 (d, J=8.7 Hz, 2H), 3.36 (s,1H) ppm. ¹³C NMR (75 MHz, CDCl₃) δ 147.3, 132.8, 128.7, 123.4, 53.3 ppm.1-Nitro-4-(1-pentynyl)benzene (4b) was obtained as a brown oil (148 mg,65%); ¹H-NMR (300 MHz, CDCl₃) δ 8.15 (d, J=8.7 Hz, 2H), 7.52 (d, J=9.0Hz, 2H), 2.42 (t, 2H), 1.68-1.61 (m, 2H), 1.05 (t, 3H) ppm. ¹³C NMR (75MHz, CDCl₃) δ 132.1, 131.7, 131.1, 123.3, 96.4, 79.3, 21.7, 21.4, 13.4.ppm1-(1-Hexynyl)-4-nitrobenzene (4c) was obtained as an amber oil (241 mg,99%). ¹H-NMR (300 MHz, CDCl₃) δ 8.14 (d, J=9.3 Hz, 2H), 7.49 (d, J=8.7Hz, 2H), 2.43 (t, J=7.5 Hz, 2H), 1.64-1.42 (m, 4H), 0.94 (t, J=6.9 Hz,3) ppm. ¹³C NMR (75 MHz, CDCl₃) δ 146.4, 132.1, 131.1, 123.3, 96.6,79.1, 30.3, 21.9, 19.1, 13.4 ppm.1-(1-Heptynyl)-4-nitrobenzene (4d) was obtained as a brown oil (234.6mg, 90%). ¹H-NMR (300 MHz, CDCl₃) δ ppm 8.13 (d, J=9.0 Hz, 2H), 7.49 (d,J=9.0 Hz, 2H), 2.41 (t, J=6.9 Hz, 2H), 1.65-1.55 (m, 2H), 1.46-1.28 (m,4H), 0.90 (t, J=7.2 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ146.4, 132.1,131.1, 123.3, 96.7, 79.1, 31.0, 27.9, 22.1, 19.4, 13.8.1-(1-Decynyl)-4-nitrobenzene (4e) was obtained as a yellow oil (131 mg,42%); ¹H-NMR (300 MHz, CDCl₃) δ 8.14 (d, J=8.7 Hz, 2H), 7.50 (d, J=8.4Hz, 2H), 2.42 (t, J=7.2 Hz, 2H), 1.63-1.58 (m, 2H), 1.43 (m, 2H), 1.28(m, 8H), 0.87 (t, J=6.3 Hz, 3H) ppm. ¹³C NMR (75 MHz, CDCl₃) δ 146.4,132.1, 131.1, 123.3, 96.7, 79.1, 31.7, 29.0, 28.9, 28.8, 28.2, 22.5,19.4, 13.9 ppm.1-(1-Dodecynyl)-4-nitrobenzene (4f).Obtained as a brown oil (216 mg, 63%); ¹H-NMR (300 MHz, CDCl₃) δ 8.14(d, J=9.0 Hz, 2H), 7.50 (d, J=8.7 Hz, 2H), 2.42 (t, J=6.9 Hz, 2H),1.63-1.55 (m, 2H), 1.45-1.40 (m, 2H), 1.25 (m, 12H), 0.86 (t, J=6.9 Hz,3H) ppm. ¹³C NMR (75 MHz, CDCl₃) δ 146.4, 132.1, 131.1, 123.3, 96.7,79.1, 31.8, 29.4, 29.2, 29.0, 28.8, 28.3, 22.5, 19.4, 14.0 ppm.1-Nitro-4-(tetradec-1-yn-1-yl)benzene (4g) was obtained as a yellowsolid (344 mg, 99%); ¹H-NMR (300 MHz, CDCl₃) δ 8.13 (d, J=9.0 Hz, 2H),7.48 (d, J=9.0 Hz, 2H), 2.41 (t, J=6.9 Hz, 2H), 1.62-1.57 (m, 2H),1.44-1.38 (m, 2H), 1.24 (m, 16H), 0.86 (t, J=6.9 Hz, 3H) ppm. ¹³C NMR(75 MHz, CDCl₃) δ 146.3, 132.0, 131.1, 123.3, 96.6, 79.1, 31.8, 29.5,29.4, 29.3, 29.0, 28.8, 28.3, 22.6, 19.4, 13.9 ppm.1-(4-Methylpent-1-ynyl)-4-nitrobenzene (4h) was obtained as a brown oil(187 mg, 77%); ¹H-NMR (300 MHz, CDCl₃) δ 8.14 (d, J=9.0 Hz, 2H), 7.50(d, J=8.7 Hz, 2H), 2.33 (d, J=6.3 Hz, 2H), 1.96-1.87 (m, J=6.6 Hz, 1H),1.04 (d, J=6.6 Hz, 6H) ppm. ¹³C NMR (75 MHz, CDCl₃) δ 146.4, 132.1,131.1, 123.3, 95.6, 80.1, 28.5, 27.9, 21.9 ppm.1-(5-Methylhex-1-ynyl)-4-nitrobenzene (4i) was obtained as a light brownoil (232 mg, 87%); ¹H-NMR (300 MHz, CDCl₃) δ 8.15 (d, J=8.4 Hz, 2H),7.51 (d, J=7.5 Hz, 2H), 2.44 (t, 2H), 1.76-1.70 (m, 1H), 1.55-1.48 (m,2H), 0.95 (d, J=6.6 Hz, 6H) ppm. ¹³C NMR (75 MHz, CDCl₃) δ 146.4, 132.1,131.1, 123.3, 96.7, 79.1, 37.2, 27.2, 22.0, 17.5 ppm.1-(3,3-Dimethylbut-1-ynyl)-4-nitrobenzene (4j) was obtained as a brightyellow solid (91.1 mg, 37%); ¹H-NMR (300 MHz, CDCl₃) δ 8.23 (d, J=7.8Hz, 1H), 8.16 (d, J=7.5 Hz, 1H), 7.50-7.46 (dd, J=3.3, 7.8, 4.2 Hz, 2H),1.34 (s, 9H) ppm. ¹³C NMR (100 MHz, CDCl₃) δ 142.7, 131.7, 125.5, 122.0,100.9, 79.1, 30.8, 30.7, 28.0 ppm.1-Nitro-4-(phenylethynyl)benzene (4k) was obtained as a bright yellowsolid (239 mg, 89%); ¹H-NMR (300 MHz, CDCl₃) δ 8.20 (d, J=8.7 Hz, 2H),7.65 (d, J=8.4 Hz, 2H), 7.57-7.54 (dd, J=6.3, 1.8, 3.3 Hz, 2H),7.40-7.37 (m, 3H) ppm. ¹³C NMR (75 MHz, CDCl₃) δ 146.8, 132.1, 131.7,129.2, 128.5, 123.5, 122.0, 94.6, 87.5 ppm.1-(Cyclohexylethynyl)-4-nitrobenzene (41) was obtained as a brown oil(284 mg, 99%); ¹H-NMR (300 MHz, CDCl₃) δ 8.11 (d, J=9.0 Hz, 2H), 7.48(d, J=7.5 Hz, 2H), 2.63-2.54 (m, 1H), 1.87-1.82 (m, 2H), 1.75-1.67 (m,2H), 1.55-1.46 (m, 2H), 1.39-1.22 (m, 4H) ppm. ¹³C NMR (75 MHz, CDCl₃) δ146.3, 132.1, 131.1, 123.3, 100.5, 79.1, 32.9, 29.6, 25.6, 24.7 ppm.

General Procedure for Hydrogenation of 4a-m.

In a 100 mL round bottom flask, alkyne 4 (1.24 mmol) was dissolved inEtOH (6 mL), PtO₂, 83% Pt (40 mg, 0.18 mmol) was added, the solvent wasdegassed under reduced pressure and hydrogen gas was introduced byballoon. The reaction was stirred at for 2 h and filtered throughCelite. The Celite pad was washed with CH₂Cl₂, EtOAc and MeOH, thefiltrate was concentrated under reduced pressure and purified by flashchromatography using 5% EtOAc-hexanes to afford the aniline 5.

4-Ethylaniline (5a) was obtained as a colorless solid (110 mg, 46%);¹H-NMR (300 MHz, CDCl₃) δ 7.05 (d, J=8.4 Hz, 2H), 6.68 (d, J=8.4 Hz,2H), 3.80 (br s, 2H), 2.58 (q, J=7.5 Hz, 2H), 1.24 (t, J=7.5 Hz, 3H)ppm. ¹³C NMR (75 MHz, CDCl₃) δ 144.0, 134.3, 128.5, 115.2, 27.9, 15.9ppm.4-Pentylaniline (5b) was obtained as a yellow brown oil (35./2 mg, 98%);¹H-NMR (300 MHz, CDCl₃) δ 6.99 (d, J=8.4 Hz, 2H), 6.64 (d, J=8.4 Hz,2H), 3.63 (br s, 2H), 2.53 (t, J=8.1 Hz, 2H), 1.62-1.52 (m, J=6.9 Hz,2H), 1.34-1.27 (m, 4H), 0.89 (t, J=7.2 Hz, 3H) ppm. ¹³C NMR (75 MHz,CDCl₃) δ 143.9, 133.5, 129.0, 115.1, 34.9, 31.4, 22.5, 13.9 ppm.4-Hexylaniline (5c) was obtained as a pale amber oil (120 mg, 81%);¹H-NMR (300 MHz, CDCl₃) δ 7.04 (d, J=8.4 Hz, 2H), 6.67 (d, J=8.4 Hz,2H), 3.52 (s, 2H), 2.56 (t, J=8.1 Hz, 2H), 1.64-157 (m, 2H), 1.42-1.33(m, 6H), 0.95 (t, J=6.6 Hz, 3H) ppm. ¹³C NMR (75 MHz, CDCl₃) δ 143.9,133.0, 129.0, 115.1, 35.0, 31.7, 28.9, 22.6, 14.1 ppm.4-Heptylaniline (5d) was obtained as a pale brown oil (139 mg, 99%);¹H-NMR (300 MHz, CDCl₃) δ 7.02 (d, J=7.8 Hz, 2H), 6.66 (d, J=8.1 Hz,2H), 3.47 (br s, 2H), 2.54 (t, J=8.1 Hz, 2H), 1.63-1.55 (m, 2H),1.35-1.28 (m, 8H), 0.93 (t, J=7.5 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ143.9, 133.0, 129.0, 115.1, 35.0, 31.8, 29.2, 22.6, 14.0 ppm.4-Decylaniline (5e) was obtained as a yellow solid crystals (145 mg,99%); ¹H-NMR (300 MHz, CDCl₃) δ 7.00 (d, J=7.8 Hz, 2H), 6.65 (d, J=8.4Hz, 2H), 3.63 (broad s, 2H), 2.51 (t, J=8.1 Hz, 2H), 1.59-1.55 (m, 2H),1.28 (s, 14H), 0.91 (t, J=6.9 Hz, 3H) ppm. ¹³C NMR (75 MHz, CDCl₃) δ143.9, 133.0, 129.0, 115.1, 35.0, 31.8, 29.5, 29.2, 22.6, 14.0 ppm.4-Dodecylaniline (5f) was obtained as a yellow oil (198 mg, 99%); ¹H-NMR(300 MHz, CDCl₃) δ 7.01 (d, J=7.5 Hz, 2), 6.65 (d, J=8.1 Hz, 2H), 3.59(broad s, 2H), 2.54-2.50 (t, J=6.9 Hz, 2H), 1.58 (m, 2H), 1.29 (m, 16H),0.92 (t, 3H) ppm. ¹³C NMR (75 MHz, CDCl₃) δ143.9, 133.0, 129.0, 115.1,35.0, 31.9, 31.8, 29.6, 29.3, 22.6, 14.1 ppm.4-Tetradecylaniline (5g) was obtained as a pale gray solid (250 mg,71%); ¹H-NMR (300 MHz, CDCl₃) δ 7.03 (d, J=7.8 Hz, 2H), 6.67 (d, J=8.1Hz, 2H), 3.55 (br s, 2H), 2.55 (t, J=7.8 Hz, 2H), 1.63-1.58 (m, 2H),1.32 (m, 22H), 0.95 (t, J=7.2 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 143.9,133.0, 129.0, 115.1, 35.0, 31.9, 31.8, 29.6, 29.5, 29.4, 29.3, 22.6,14.1.4-(4-Methylpentyl)aniline (5h) was obtained as a colorless solid (176mg, 99%); ¹H-NMR (300 MHz, CDCl₃) δ 7.04 (d, J=7.8 Hz, 2H), 6.68 (d,J=8.1 Hz, 2H), 3.60 (broad s, 2H), 2.54 (t, J=7.8 Hz, 2H), 1.64-1.57 (m,3H), 1.30-1.23 (m, J=8.4 Hz 2H), 0.95 (d, J=7.2 Hz, 6H) ppm. ¹³C NMR (75MHz, CDCl₃) δ 144.8, 133.0, 129.1, 115.1, 38.6, 35.3, 29.6, 27.8, 22.6ppm.4-(5-Methylhexyl)aniline (5i) was obtained as an orange oil (173 mg,86%); ¹H-NMR (300 MHz, CDCl₃) δ 7.05 (d, J=8.4 Hz, 2H), 6.68 (d, J=8.1Hz, 2H), 3.61 (br s, 2H), 2.57 (t, J=7.8 Hz, 2H), 1.56-1.66 (m, 3H),1.35-1.44 (m, 2H), 1.23-1.30 (m, 2H), 0.95 (d, J=6.3 Hz, 6H) ppm. ¹³CNMR (75 MHz, CDCl₃) δ 143.9, 133.0, 129.1, 115.2, 38.8, 35.1, 32.1,27.9, 27.0, 22.6 ppm.4-(3,3-Dimethylbutyl)aniline (5j) was obtained as a brown solid (51 mg,64%); ¹H-NMR (300 MHz, CDCl₃) δ 7.00 (d, J=7.8 Hz, 2H), 6.64 (d, J=8.4Hz, 2H), 3.47 (br s, 2H), 2.41-2.50 (m, 2H), 1.43-1.49 (m, 2H), 0.96 (s,9H) ppm. ¹³C NMR (75 MHz, CDCl₃) δ 143.8, 133.5, 128.9, 115.2, 46.6,30.4, 30.1, 29.6, 29.3 ppm.4-(2-Phenylethyl)aniline (5k) was obtained as a pale yellow solid (192mg, 91%); ¹H-NMR (300 MHz, CDCl₃) δ 7.42 (t, J=7.5, 7.2 Hz, 2H). 7.33(t, J=4.2, 7.5 Hz, 3H), 7.12 (d, J=7.8 Hz, 2H), 6.73 (d, J=7.8 Hz, 2H),3.59 (br s, 2H), 2.94-3.04 (m, 4H) ppm. ¹³C NMR (75 MHz, CDCl₃) δ144.4,142.1, 131.8, 129.2, 128.6, 128.4, 125.8, 115.3, 38.4, 37.2 ppm.4-(2-Cyclohexylethyl)aniline (5I) was obtained as a brown oil (261 mg,99%); ¹H-NMR (300 MHz, CDCl₃) δ 7.06 (d, J=7.8 Hz, 2H), 6.68 (d, J=8.4Hz, 2H), 3.60 (br s, 2H), 2.59 (t, 2H), 1.76-1.86 (m, 5H), 1.50-1.57 (m,2H), 1.21-1.36 (m, 4H), 0.95-1.06 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ143.8, 133.3, 129.0, 115.3, 39.7, 37.2, 33.3, 32.3, 26.7, 26.3 ppm.

General Procedures for the Synthesis of Analogues 2a-m. Method A:

Aniline 5 (0.80 mmol) was dissolved in CH₂Cl₂ (8.0 mL) and sulfonylchloride 6 (0.80 mmol) was added slowly, followed by the slow additionof NEt₃ (0.40 mmol). The resultant mixture was stirred for 15 to 18hours, diluted with water (5 mL) and washed with CH₂Cl₂ (3×5 mL). Thecombined organic layers were washed with brine (5 mL) and H₂O (5 mL),dried over Na₂SO₄ and concentrated under reduced pressure.The residue was dissolved in THF-MeOH—H₂O (1:1:1) and crushed pellets ofNaOH (20.0 mg, 0.50 mmol) were added to the stirring solution. After 18h, the reaction mixture was concentrated under reduced pressure, dilutedwith H₂O (5 mL) and washed with EtOAc (3×5 mL). The combined organiclayers were washed with water (5 mL), dried over Na₂SO₄ and concentratedunder reduced pressure. The products were purified by preparativereverse phase HPLC.

Method B:

To an aqueous NaHCO₃ solution (2.0 mL, 5.0 M, 0.44 mmol) was added theaniline 5 (0.22 mmol) and 1,4-dioxane (1.0 mL). The biphasic mixture wasstirred vigorously at 0° C. and a solution of sulfonyl chloride 6 (54.0mg, 0.22 mmol) in 1,4-dioxane (1 mL) was added to the reaction dropwiseat 0° C. The reaction was warmed to room temperature and stirred for 18h, after which the reaction mixture was concentrated under reducedpressure, diluted with H₂O (5 mL) and extracted with EtOAc (3×5 mL). Thecombined organic layers were washed with brine (5 mL) and H₂O (5 mL),dried over Na₂SO₄ and concentrated under vacuum.The crude product was dissolved in THF-MeOH—H₂O (1:1:1) (3 mL) andcrushed pellets of NaOH (20.0 mg, 0.50 mmol) were added to the stirringsolution. After 18 h, the reaction mixture was concentrated underreduced pressure, diluted with H₂O (5 mL) and extracted with EtOAc (3×5mL). The combined organic layers were washed with H₂O (5 mL), dried overNa₂SO₄ and concentrated under reduced pressure. The crude product waspurified by preparative reverse phase HPLC.5-[N-(4-butylphenyl)sulfamoyl]-2-hydroxybenzoic acid (1), synthesizedusing Method A, was obtained as a colorless powder (99.4 mg, 71%);¹H-NMR (300 MHz, CD₃OD) δ 8.28 (d, J=2.4 Hz, 1H), 7.58-7.54 (dd, J=2.4,9.0, 2.1 Hz, 1H), 7.02-6.95 (m, 4H), 6.81 (d, J=9.0 Hz, 1H), 2.52 (t,2H), 1.56-1.46 (m, 2H), 1.32-1.25 (m, 2H), 0.92 (t, 3H) ppm. ¹³C NMR(100 MHz, CD₃OD) δ 173.9, 166.7, 140.6, 136.5, 132.9, 131.7, 130.1,129.9, 129.6, 122.9, 119.4, 117.8, 35.8, 34.7, 23.2, 14.2 ppm.5-[N-(4-ethylphenyl)sulfamoyl]-2-hydroxybenzoic acid (2a), synthesizedusing Method A, was obtained as an off-white powder (69.6 mg, 62%);¹H-NMR (300 MHz, CD₃OD) δ 8.28 (d, J=2.1 Hz, 1H), 7.57 (dd, J=2.7, 8.7,3.0 Hz, 1H), 6.95-7.03 (m, 5H), 6.81 (d, J=9.0 Hz, 1H), 2.51 (q, 2H),1.91 (s, 1H), 1.12 (t, 3H) ppm. ¹³C NMR (100 MHz, CD₃OD) δ 180.7, 174.3,142.2, 136.3, 132.7, 131.7, 129.4, 123.0, 120.1, 117.9, 108.9, 30.1,29.1, 24.3, 16.1 ppm.2-hydroxy-5-[N-(4-propylphenyl)sulfamoyl]benzoic acid (2b), synthesizedusing Method A, was obtained as a pink powder (178 mg, 19%); ¹H-NMR (300MHz, CD₃OD) δ 8.17 (d, J=2.4 Hz, 1H), 7.77 (dd, J=2.7, 8.7, 2.7 Hz, 1H),7.00 (dd, J=8.7, 11.7, 8.7 Hz, 4H), 6.98 (d, 1H) ppm. ¹³C NMR (100 MHz,CD₃OD) δ 172.3, 166.2, 140.9, 136.3, 134.8, 131.6, 131.3, 130.1, 123.2,118.9, 113.8, 38.2, 25.5, 13.9 ppm.2-hydroxy-5-[N-(4-pentylphenyl)sulfamoyl]benzoic acid (2c), synthesizedusing Method B, was obtained as a pale orange powder (83.7 mg, 99%);¹H-NMR (300 MHz, CD₃OD) δ 8.29 (d, J=2.4 Hz, 1H), 7.51 (dd, J=2.1, 8.7,2.4 Hz, 1H), 6.95-7.02 (m, 5H), 6.77 (d, J=8.7 Hz, 1H), 2.50 (t, 2H),1.51-1.56 (m, 2H), 1.29-1.34 (m, 4H), 0.87 (t, 3H) ppm. ¹³C NMR (100MHz, CD₃OD) δ 174.0, 166.8, 140.6, 136.6, 132.6, 131.6, 129.8, 129.3,122.9, 120.2, 117.6, 36.1, 32.5, 32.2, 30.7, 23.4, 14.3 ppm.5-[N-(4-hexylphenyl)sulfamoyl]-2-hydroxybenzoic acid (2d), synthesizedusing Method B, was obtained as an off white powder (54.7 mg, 60%);¹H-NMR (300 MHz, CD₃OD) δ 8.30 (d, J=2.4 Hz, 1H), 7.51-7.55 (2.1, 8.7,2.4 Hz, 1H), 6.95-7.02 (m, 4H), 6.78 (d, J=8.7 Hz, 1H), 2.49 (t, 2H),1.52 (m, 2H), 1.27 (m, 6H), 0.86 (t, 3H) ppm. ¹³C NMR (100 MHz, CD₃OD) δ180.6, 174.4, 166.9, 140.7, 136.7, 132.8, 131.8, 130.1, 129.5, 123.0,120.3, 117.9, 36.3, 32.9, 32.7, 30.1, 23.8, 14.5 ppm.5-[N-(4-heptylphenyl)sulfamoyl]-2-hydroxybenzoic acid (2e), synthesizedusing Method B, was obtained as a colorless powder (58.0 mg, 46%);¹H-NMR (300 MHz, CD₃OD) δ 8.30 (d, J=3.0 Hz, 1H), 7.51 (dd, J=2.4, 8.7,2.4 Hz, 1H), 6.94-7.02 (m, 5H), 6.77 (d, J=9.0 Hz, 1H), 2.48 (t, 2H),1.52 (m, 2H), 1.26 (m, 8H), 0.87 (t, 3H) ppm. ¹³C NMR (100 MHz, CD₃OD) δ180.3, 174.2, 166.8, 140.6, 136.5, 132.6, 131.7, 129.9, 129.4, 123.2,122.8, 120.1, 117.7, 36.1, 32.9, 32.5, 30.2, 23.6, 14.4 ppm.5-[N-(4-decylphenyl)sulfamoyl]-2-hydroxybenzoic acid (2f), synthesizedusing Method B, was obtained as a colorless powder (74.5 mg, 61%);¹H-NMR (300 MHz, CD₃OD) δ 8.31 (d, J=2.4 Hz, 1H), 7.56-7.53 (dd, J=2.4,9, 2.1 Hz, 1H), 6.95-7.01 (m, 4H), 6.79 (d, J=8.4 Hz, 1H), 3.34 (s, 1H),2.50 (t, 2H), 1.53 (m, 2H), 1.25 (m, 14H), 0.89 (t, 3H) ppm. ¹³C NMR(100 MHz, CD₃OD) δ 174.2, 166.8, 140.6, 136.5, 132.7, 131.7, 129.9,129.5, 122.8, 119.9, 117.8, 36.2, 33.0, 32.5, 30.6, 30.5, 30.4, 30.2,23.7, 14.4. ppm5-[N-(4-dodecylphenyl)sulfamoyl]-2-hydroxybenzoic acid (2g), synthesizedusing Method B, was obtained as a colorless powder (140 mg, 40%); ¹H-NMR(300 MHz, CD₃OD) δ 8.30 (s, 1H), 7.56 (d, J=7.8 Hz, 1H), 6.95-7.02 (m,J=8.4, 9 Hz, 5H), 6.80 (d, J=8.4 Hz, 1H), 2.51 (t, 2H), 1.52 (m, 2H),1.27 (m, 16H), 0.94 (t, 3H) ppm. ¹³C NMR (75 MHz, CD₃OD) δ 167.02,140.9, 136.7, 132.9, 131.9, 130.1, 129.7, 123.0, 118.0, 36.4, 33.3,32.8, 30.9, 30.7, 30.5, 23.9, 14.6 ppm.2-hydroxy-5-[N-(4-tetradecylphenyl)sulfamoyl]benzoic acid (2h),synthesized using Method B, was obtained as a colorless powder (98.2 mg,72%); ¹H-NMR (300 MHz, CD₃OD) δ 8.31 (d, J=2.1 Hz, 1H), 7.53 (dd, J=2.4,8.7 Hz, 1H), 6.95-7.03 (m, 5H), 6.77 (d, J=8.7 Hz, 1H) ppm. ¹³C NMR (75MHz, CD₃OD) δ 166.9, 140.7, 136.8, 132.8, 131.9, 130.0, 129.6, 123.0,120.4, 117.8, 36.4, 33.3, 32.8, 30.9, 30.7, 30.5, 23.9, 14.7 ppm.2-hydroxy-5-[N-(4-(4-methylpentyl)phenyl]sulfamoyl)benzoic acid (2i),synthesized using Method B, was obtained as a colorless powder (79.1 mg,43%); ¹H-NMR (300 MHz, CD₃OD) δ 8.31 (d, J=2.4 Hz, 1H), 7.54 (dd, J=2.4,8.7 Hz, 1H), 6.98-7.01 (m, 5H), 6.79 (d, J=8.4 Hz, 1H), 2.49 (t, 2H),1.57 (m, 3H), 1.17 (m, 2H), 0.84 (t, 6H) ppm. ¹³C NMR (100 MHz, CD₃OD) δ166.8, 140.6, 136.5, 132.6, 131.7, 129.9, 129.4, 122.8, 120.1, 117.7,117.1, 39.6, 36.4, 30.8, 30.3, 28.9, 22.9 ppm.2-hydroxy-5-[N-(4-(5-methylhexyl)phenyl)sulfamoyl]benzoic acid (2j),synthesized using Method B, was obtained as an off-white powder (231 mg,82%); ¹H-NMR (300 MHz, CD₃OD) δ 8.34 (d, J=2.4 Hz, 1H), 7.57 (dd, J=2.4,8.7 Hz, 1H), 6.98 (m, 4H), 6.80 (d, J=8.7 Hz, 1H), 2.49 (t, 2H), 1.93(s, 1H), 1.41-1.50 (m, 3H), 1.09-1.26 (m, 5H), 0.82 (d, 6H) ppm. ¹³C NMR(75 MHz, CD₃OD) δ 174.5, 166.9, 140.7, 136.6, 132.8, 131.9, 130.1,129.5, 123.0, 120.3, 118.1, 40.1, 36.4, 32.9, 29.2, 28.2, 23.2 ppm.5-[N-(4-(3,3-dimethylbutyl)phenyl)sulfamoyl]-2-hydroxybenzoic acid (2k),synthesized using Method A, was obtained as an off-white powder (27.4mg, 43%); ¹H-NMR (300 MHz, CD₃OD) δ 8.29 (s, 1H), 7.53 (d, J=6 Hz, 1H),6.91-7.02 (m, 5H), 6.80 (d, J=9.9 Hz, 1H), 2.45-2.51 (m, 2H), 1.38-1.44(m, 2H), 0.85-0.99 (m, 1H), 0.93 (s, 9H) ppm. ¹³C NMR (75 MHz, CD₃OD) δ166.9, 141.3, 136.5, 132.7, 131.8, 130.0, 129.9, 129.7, 129.5, 123.7,123.1, 122.9, 47.4, 31.5, 31.2, 29.6 ppm.2-hydroxy-5-[N-(4-phenethylphenyl)sulfamoyl]benzoic acid (2I),synthesized using

Method B, was obtained as a colorless powder (75.1 mg, 53%); ¹H-NMR (300MHz, CD₃OD) δ 8.31 (d, J=2.4 Hz, 1H), 7.55 (dd, J=2.4, 8.4, 2.4 Hz, 1H),7.02-7.20 (m, 5H), 6.95 (m, 4H), 6.80 (d, J=8.4 Hz, 1H), 2.77 (s, 4H)ppm. ¹³C NMR (75 MHz, CD₃OD) δ 180.4, 174.2, 166.8, 142.7, 139.5, 136.8,132.6, 131.7, 130.1, 129.9, 129.5, 129.3, 129.1, 126.8, 122.7, 120.1,117.8, 38.9, 38.3 ppm.

5-[N-(4-(2-cyclohexylethyl)phenyl)sulfamoyl]-2-hydroxybenzoic acid (2m),synthesized using Method B, was obtained as a colorless powder (135 mg,45%); ¹H-NMR (300 MHz, CD₃OD) δ 8.33 (d, J=2.4 Hz, 1H), 7.56 (dd, J=2.7,8.7, 2.4 Hz, 1H), 6.90-6.99 (m, 4H), 6.80 (d, J=9 Hz, 1H), 2.45-2.51 (m,2H), 1.64-1.72 (m, 5H), 1.33-1.42 (m, 2H), 1.10-1.26 (m, 4H), 0.85-0.93(m, 2H) ppm. ¹³C NMR (75 MHz, CD₃OD) δ 174.5, 166.9, 141.0, 136.6,132.8, 131.9, 130.1, 129.6, 123.0, 120.3, 118.0, 40.6, 38.6, 34.6, 33.7,27.9, 27.6 ppm.Methyl 5-[N-(4-butylphenyl)sulfamoyl]-2-hydroxybenzoate (7). Isolated asan intermediate in the synthesis of 1, ester 7 was obtained as acolorless solid (618 mg, 90% yield). ¹H-NMR (300 MHz, CDCl₃) δ 8.30 (d,J=2.4 Hz, 1H), 7.82 (dd, J=8.7, 2.4 Hz, 1H), 6.97-7.00 (m, 4H), 6.96 (d,J=9.0 Hz, 1H), 3.88 (s, 3H), 2.49 (t, 2H), 1.47-1.52 (m, 2H), 1.21-1.31(m, 2H), 0.87 (t, 3H) ppm. ¹³C NMR (75 MHz, CDCl₃) δ 169.4, 164.5,140.3, 133.9, 133.7, 130.2, 129.6, 129.1, 122.1, 118.4, 112.1, 52.6,34.8, 33.3, 22.1, 13.7 ppm.Methyl 2-(2-amino-2-oxoethoxy)-5-(N-(4-butylphenyl)sulfamoyl)benzoate(8). Ester 7 (311 mg, 0.86 mmol) and anhydrous Cs₂CO₃ (1.04 g, 4.28mmol) were added to a solution of 2-iodoacetamide (161 mg, 1.72 mmol) inanhydrous acetone (6 mL). The reaction mixture was refluxed withvigorous stirring for 18 h. After cooling to room temperature, thereaction mixture was filtered and the filtrate evaporated under reducedpressure. The residue was dissolved in EtOAc (5 mL) and the solution waswashed with 1N HCl (2×5 mL), 0.5 N Na₂CO₃ (5 mL) and water (5 mL), driedover Na₂SO₄ and concentrated under reduced pressure. The crude productwas purified by flash column chromatography with 60% EtOAc-hexanes,affording product 8 as a colorless powder (114 mg, 31% yield). ¹H-NMR(300 MHz, CDCl₃) δ 11.30 (s, 1H), 8.14 (d, J=2.4 Hz, 1H), 7.48 (dd,J=2.4, 9.0 Hz, 1H), 7.13 (d, J=8.4 Hz, 1H), 7.01-6.96 (m, 4H), 6.62 (brs, 1H), 5.90 (br s, 1H), 4.23 (s, 2H), 2.58 (t, 2H), 1.51-1.61 (m, 2H),1.28-1.37 (m, 2H), 0.92 (t, 3H) ppm. ¹³C NMR (75 MHz, CDCl₃) δ 170.3,165.0, 143.6, 136.7, 134.6, 130.7, 129.4, 127.4, 118.5, 112.3, 54.4,52.8, 35.0, 33.3, 22.2, 13.8 ppm.5-[N-(4-butylphenyl)sulfamoyl]-2-(carbamoylmethoxy)benzoic acid (3).Ether 8 (43 mg, 0.10 mmol) was dissolved in 1:1:1 THF-MeOH—H₂O (9 mL)and crushed pellets of NaOH (107 mg, 2.68 mmol) were added to thestirring solution. After stirring for 18 h, the reaction mixture wasconcentrated under reduced pressure, diluted with water (5 mL) andwashed with EtOAc (3×5 mL). The combined organic layers were washed withbrine (5 mL), water (5 mL) and dried over Na₂SO₄ and concentrated undervacuum. Compound 3 (40.0 mg) was obtained in 98% yield as an off-whitepowder. ¹H-NMR (300 MHz, CDCl₃) δ 8.02 (d, J=2.4 Hz, 1H), 7.64 (dd,J=2.4, 9.0, 2.1 Hz, 1H), 7.07-7.16 (m, 4H), 7.04 (d, J=8.7 Hz, 1H), 4.24(s, 2H), 2.60 (t, 2H), 1.52-1.63 (m, 2H), 1.29-1.39 (m, 2H), 0.93 (t,3H) ppm. ¹³C NMR (100 MHz, CDCl₃) δ 173.0, 172.9, 166.7, 144.5, 138.6,135.6, 132.4, 130.1, 129.2, 119.0, 114.5, 54.7, 36.0, 34.6, 23.2, 14.1ppm.

Isolation of HMG CoA Reductase

A clone containing the HMG-CoA reductase gene from S. aureus (mvaA) in apET28 vector (Novagen) with an N-terminal 6×-His tag was transformedinto competent BL21 [DE3] cells grown to an OD of 0.6 and induced with1.0 mM of IPTG. After 3 h of induction at 37° C. the cells were lysedwith a French press. The resulting lysate was clarified bycentrifugation at 50,000 rpm for 1 h, and the overexpressed proteinisolated from the supernatant using a Talon column (Clonetech). Afterconcentration, the protein was further purified using Sephacryl S200(HiPrep 26/60 Pharmacea Biotech) equilibrated with the reaction buffer.The final protein solution was concentrated to 10 mg/ml forcrystallization trials and subsequently diluted to 1 mg/ml for kineticexperiments.

Identification of Class II HMG-CoA Reductase Inhibitors

The kinetic studies were carried out in Corning 3695 96-well formatplates with each experiment occupying a 6×4 grid of wells. Each of thewells contained a total volume of 200 μl of a solution containingTris-HCl (100 mM), K2HPO4 (25 mM) and KCl (50 mM) at a pH of 7.5.HMG-CoA and NADPH were added to each well to achieve finalconcentrations of 150 at 300 μM, respectively. Reaction was initiated bythe addition of purified HMGR (50 ng) using a multi-channel pipette andwas followed by measuring the absorbance at 340 nm for 3 min at 10 sintervals using a BioTex Synergy H1 plate reader. The slope of thelinear portion of this curve was taken as the rate, measured inmilliOD/min.

Inhibitor was added to the wells at five different concentrations withfourfold redundancy. No inhibitor was added to one column of wells thatserved as a 100% control. The concentrations of inhibitor used wereadjusted to bracket the 50% inihibition level, ensuring minimal errorsbetween data points. The four redundant readings were averaged and thevariation calculated to give a standard deviation. These values weregraphed and the final IC₅₀ and its error bars determined.

Inhibition of HMG CoA Reductase and Bactericidal Activity

Inhibition of purified S. aureus HMGR by 1,2a-m and 3 was assayed inparallel format by monitoring oxidation of NADPH to NADP+ in theconversion of HMG CoA to mevalonate by monitoring change in absorbanceat 340 nm using a 96-well plate reader. All experiments were performedin quadruplicate with IC₅₀ values determined by curve fitting. The IC₅₀values of 1 and its analogues 2a-m and 3 are shown in Table 1.

In parallel with the inhibition studies, all molecules were also testedfor the ability to inhibit growth of MRSA in culture. The minimuminhibitory concentration (MIC) and the minimum bactericidalconcentration (MBC) of the HMG-CoA reductase inhibitors were tested intriplicate against methicillin-resistant S. aureus (MRSA) in a microdilution method, according to Clinical and Laboratory Standards (CLSI)(Table 1). Similar tests against E. coli, which has the alternativeMEP/DOXP isoprenoid biosynthesis pathway and therefore lacks HMGR, showno inhibitory effects of any of the compounds at the concentrationstested.

TABLE 1 Inhibition of S. aureus HMGR and Bactericidal Activity AgainstMRSA of Designed Analogues. MBC, Analog R^(a) cLogP^(b) IC₅₀, μM MIC, μMμM 1 C₄H₉ 3.14 91 ± 5 ^(c) ^(c) 2a C₂H₅ 2.22 ^(d) ^(c) ^(c) 2b C₃H₇ 2.68^(d) ^(c) ^(c) 2c C₅H₁₁ 3.61 98 ± 3 ^(c) ^(c) 2d C₆H₁₃ 4.07 77 ± 6 128128 2e C₇H₁₅ 4.54   28 ± 2.0 64 64 2f C₁₀H₂₁ 5.93  7.2 ± 0.6 16 16 2gC₁₂H₂₅ 6.86  5.9 ± 0.5 128 128 2h C₁₄H₂₉ 7.79  1.9 ± 0.15 ^(c) ^(c) 2i(CH₂)₃CH(CH₃)₂ 3.95 36 ± 4 64 ^(e) 2j (CH₂)₄CH(CH₃)₂ 4.41   22 ± 1.0 6464 2k (CH₂)₂C(CH₃)₃ 4.00 ^(d) ^(c) ^(c) 2l (CH₂)₂c-C₆H₁₁ 3.90 16.2 ± 1.032 32 2m (CH₂)₂Ph 3.49 ^(d) ^(c) ^(c) 3 C₄H₉ ^(c) 1.88 ^(d) ^(c) ^(c)^(a)Structures of 2a-m and 3 are shown in FIG. 3. ^(b)cLogP calculationswere performed using OSIRIS Molecular Property calculator of Actelionlabs. ^(c)No growth inhibition of MRSA was noted at any of theconcentrations tested. ^(d)No significant inhibition of HMGR activitywas observed at the concentrations tested. ^(e)Analogue 2i displayedbacteriostatic activity but no bactericidal activity.Inhibition of HMGR increased with increasing length of the alkyl “tail”,and hence with increasing lipophilicity. Conversely, truncation of thetail resulted in the loss of enzymatic inhibition. The C₁₄ tail of 2hafforded the greatest inhibition of HMGR (IC₅₀=1.9 μM), a 45-foldimprovement over lead compound 1. However, when the bactericidalactivity of these compounds was examined, it was found that activity wasobserved only for those compounds with a tail of C₆ or longer; moreover,whereas the C₁₀ analogue 2f exhibited an MBC of 16 μM—nearly comparableto its IC₅₀ of 7.2 μM—the analogues with longer tails (2g and 2h) lostbactericidal activity. Differences between enzymatic and bactericidalactivity of these analogues may be attributable to the ability of thesecompounds to traverse the plasma membrane. The most lipophilic members2g and 2h may lose activity owing to their localization within themembrane, thus suggesting that an optimal lipophilicity might bedesirable for maximal bactericidal activity and that 2f bestapproximates that optimal value. Examination of calculated logP values(Table 1) suggests that an optimal value would lie in the range of 6-7.

The inhibitory activity of the branched-chain analogues 2i and 2jclosely resembled that of their straight-chain counterparts. The IC₅₀ of2j, with a branched-chain C₇ tail, is 22 μM, which is close to the IC₅₀of the straight-chain C₇ analogue 2e of 28 μM. Likewise, their MBCvalues are both 64 μM. On the other hand, analogue 2k, with a tailcontaining a bulky t-butyl group, failed to exhibit any activity,suggesting that the bulky t-butyl group cannot be accommodated withinthe active site of HMGR. The analogue 2l with a terminal cyclohexylsubstitutent showed activity closely parallel to that of the comparablylipophilic alkyl analogue 2j but was the second-most bactericidalanalogue. Interestingly, the terminal phenyl analogue 2m exhibited noenzymatic inhibition or bactericidal activity, perhaps indicating thatthe rigid phenyl ring couldn't be accommodated within the HMGR activesite.

The analogue 3, in which the phenol had been O-alkylated with anacetamide group designed to engage Asn213 in a cooperative hydrogenbond, failed to show any enzymatic inhibition or bactericidal activity.The lack of bactericidal activity can be explained from the low logPvalue shown in Table 1, but the lack of enzymatic inhibition by 3suggests that the alkylation of the phenol has removed thewater-mediated hydrogen bond to Asn213 without a compensatorycooperative hydrogen bond.

Evaluation of HMG-CoA Reductase Inhibitors Against MRSA, VRE, and VRSA

The MIC and MBC of HMGR inhibitors against vancomycin resistantEnterococcus faecalis (VRE) ATCC 51299 and methicillin-resistantStaphylococcus aureus (MRSA) ATCC 43300 were tested in triplicate in amicro dilution broth method, using Muller Hinton broth (MHB) and 96 wellplates, and according to CLSI. The MICS of compounds against vancomycinresistant S. aureus (VRSA) NARSA Strain ID VRS10 were tested intriplicate using a micro dilution broth method, using Tryptone Soyabroth and 96 well-plate and according to CLSI. The bacterial cultureswere incubated at 37° C. for 20 hours. The bacterial cultures wereincubated at 37° C. for 20 hours. Results are presented in Tables 2 and3.

TABLE 2 The MIC and MBC of HMG-CoA reductase inhibitors againstvancomycin resistant Enterococcus faecalis (VRE) andmethicillin-resistant Staphylococcus aureus (MRSA) VRE MRSA CompoundMIC/MBC MIC/MBC 2f 32/32 16/16 2l 64/64 32/32 2j 128/128 64/64 2d >128128/128 2e 64/64 64/64 2g 128/128  128/>128 2k 128/128 >128 2i 32/64 64/>128 2i pure  128/>128  128/>128

TABLE 3 The MIC of HMG-CoA reductase inhibitors against VRSA Compoundsname VRSA MIC (μM) 2f 16 2l 32 2j 32 2e 64 vancomycin >256

Evaluation of HMG-CoA Reductase Inhibitors Against Clinical Isolates

The MICs and MBCs of HMG-CoA reductase inhibitors against ten clinicalisolates of MRSA and vancomycin intermediate S. aureus (VISA) isolateswere tested in triplicate against 10 clinical methicillin-resistantStaphylococcus aureus (MRSA) and vancomycin intermediate Staphylococcusaureus (VISA) isolates in micro dilution broth method and according toCLSI. The bacterial cultures were incubated at 37° C. for 20 hours.Results are presented in Table 4.

TABLE 4 The MIC and MBC of HMG-CoA reductase inhibitors against clinicalMRSA and VISA isolates 2f 2l 2j 2e MIC/MBC MIC/MBC MIC/MBC MIC/MBCStrain (μM) (μM) (μM) (μM) VISA/NRS1 4/4 16/16 16/16 16/16 VancomycinIntermediate Staphylococcus aureus USA400/NRS123  4/16 16/16 16/32 32/32USA100/NRS382 8/8 16/16 16/32 32/32 USA200/NRS383 4/4 16/16 16/32 16/32USA300/NRS384 4/8 16/16 16/32 32/32 USA500/NRS385 4/8 16/16 16/16 16/16USA700/NRS386 4/8 16/16 16/16 32/32 USA800/NRS387 4/8 16/16 16/32 16/32USA1000/NRS483 4/8  8/16 16/32 16/32 USA1100/NRS484 4/8 16/16 16/3216/32

Time-Kill Assays

The bactericidal effect of compound 2f was tested against MRSA intime-kill assays. Bacterial cells in the logarithmic growth phases wereexposed to compound 2f concentrations equivalent to 1×, 2× and 3×MIC inMHB. Bacteria were cultured for 20h. Colony forming units (cfu/mL) weredetermined after 0, 2, 4, 6, 8, and 10 h of incubation. The data ispresented as the average of triplicates±standard deviations in FIG. 4.The data points without error bar indicate that standard deviation istoo small to be seen.

Treatment of MRSA with 3×MIC of compound 2f results in completeelimination of MRSA cells within a two-hour window. Treatment of MRSAwith 1×MIC or 2×MIC of compound 2f reduces the MRSA cell count moregradually, with four hours are required to eliminate bacteriacompletely. Vancomycin treatment (3×MIC) failed to reduce the number ofCFU by 3-log₁₀ within a 10 hour window. These results indicate thatcompound 2f is better than vancomycin at eliminating MRSA cells. Thisinformation is clinically relevant as it would impact the size andtiming of the dose given to patients with MRSA infections.

In Vitro Cell Viability/Cytotoxicity Studies

Cytotoxicity of the compounds was evaluated using J774A.1 cells(mitochondrial function) in the CellTiter 96® AQ_(ueous) Non-RadioactiveCell Proliferation Assay (Promega). The assay measures the bioreductionof3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) to formazan by metabolically active cells. Formazan is measured bymeasuring the absorbance at 490 nm. Briefly, ˜2×10⁴ J774A.1 cellssuspended in 200 μL DMEM supplemented with 10% fetal bovine serum (FBS),L-glutamine, NaHCO₃, pyridoxine-HCl, and 45,000 mg/L glucose andpreserved with 1% penicillin-streptomycin solution were seeded in96-well plates and incubated at 37° C. in a 5% CO₂ atmosphere. The cellswere cultured for 24 hours (60% confluency) before the assays. TheJ774A.1 cells were further incubated with 100 μM of compounds (2f, 2l,2j, and 2e) and DMSO as a control for 24 hours. The culture media wasdiscarded, and the cells in each well were washed with PBS andre-suspended with 100 μL of cell culture media prior to addition of theassay reagent. The plates were incubated for 4 hours at 37° C. in ahumidified 5% CO₂ atmosphere. The absorbance at 490 nm was recordedusing a 96-well Elisa plate reader (SoftMax Prolnc., USA). Results wereexpressed as the percentage mean absorbance by cells upon incubationwith various treatments (compounds 2f, 2j, 2i, and 2e) with respect toincubation with DMSO. The results are expressed as means from sixmeasurements±SD.

MTS assays conducted on compounds 2f, 2j, 2i, and 2e and DMSO control atconcentrations of 100 μM showed no significant differences in percentagemean absorption between various treatments (FIG. 5), indicating that thecompounds are not toxic in vitro at this concentration.

Testing Class II HMGR Inhibitors Against MRSA and VRE Using aLive-Animal Infection Model

Caenorhabditis elegans worms were infected with MRSA (1500 adults worms)and VRE (1500 adults worms) for 15 hours. Infected worms were treatedfor 16 hours with 100 μM compounds (2f, 21, and 2e) or DMSO. Vancomycin(100 μM) was used as a control for MRSA and linezolid (100 μM) was usedas a control for VRE. After treatment, infected worms were washed fivetimes with M9 buffer and 400 mg of 1.0-mm silicon carbide particles(Biospec Products, Bartlesville, Okla.) were added to each tube, thetubes were vortexed at maximum speed for one minute, which disrupts theworms but does not affect bacterial survival, and the resultingsuspension was diluted and plated on tryptic soy agar containingkanamycin to select for MRSA and VRE. The results, presented in Tables 5and 6, indicate that class II HMGR inhibitors are more effective intreating worms infected with MRSA or VRE than vancomycin or linezolid,respectively. None of the compounds affected the survival of uninfectedC. elegans.

Statistical Analysis

All statistical analyses were performed using the Student two-tailedt-test using MICROSOFT EXCEL. P values <0.05 were consideredsignificant.

TABLE 5 Treatment of C. elegans infected with MRSA Log TreatmentCFU/worm Log Reduction DMSO control 5.04 ± 0.10 0.00 2f 2.33 ± 0.092.71* 2l 2.20 ± 0.43 2.84* 2e 1.80 ± 0.10 3.24* Vancomycin 3.26 ± 0.201.78*

TABLE 6 Treatment of C. elegans infected with VRE Treatment Log CFU/wormLog Reduction DMSO control 4.90 ± 0.01 0.00 2f 0.70 ± 0.37 4.20* 2l 0.43± 0.68 4.47* 2e 0.86 ± 1.03 4.04* Linezolid 1.47 ± 0.06 3.43* Valuesrepresent the mean ± standard error numbers of CFU in triplicates.Statistical significance levels were defined as P < 0.01 (shown byasterisks).

Evaluation of HMG-CoA Reductase Inhibitors Against Other BacterialSpecies Species

Antimicrobial activities of compounds 2f and 2j were initially screenedat 100 μM concentration against Gram-positive species Bacillus anthracisSterne strain (34F2), Bacillus subtilis, Bacillus cereus, and Listeriamonocytogenes and Gram-negative species Brucella abortus. Both compoundsshowed inhibition of growth at 100 μM concentration and were testedfurther to determine the MIC and MBC in micro dilution broth method,using MHB and according to CLSI. The results are presented in Table 7.

TABLE 7 MIC and MBC for compounds 2f and 2j against bacterial species.Bacillus Bacillus Bacillus Listeria Com- anthracis subtilis cereusmonocytogenes Brucella pounds MIC/MBC MIC/MBC MIC/MBC MIC/MBC abortus 2f16/16 16/16 16/16  64/>128 128 2j 32/32 32/32 64/64 128/>128 64

Evaluation of HMG-CoA Reductase Inhibitors Against Fungal Species

Antifungal activities of 22 HMG-CoA Reductase inhibitors were initiallytested at 100 μM concentration against Candida albicans ATCC 18804. Thecompounds that showed any inhibition of growth at 100 μM concentrationwere tested further to determine the MIC and minimum fungicidalconcentration (MFC) in micro dilution broth method, using yeast mediumbroth (YM).

TABLE 8 MIC and MFC for compounds against Candida albicans Candidaalbicans Compounds MIC/MFC 2f 16/16  2j 128/>128 2e 64/128

Bactericidal Activity of HMG-CoA Reductase Inhibitors in anAdherent-Cell Biofilm

An adherent biofilm for susceptibility testing was formed using aclinical isolate of Staphylococcus aureus (ATCC 6538). S. aureusbiofilms were prepared in a tissue culture non-treated polystyrene96-well plate (Corning) and washed with phosphate-buffered saline (PBS)to remove unbound bacteria. Subsequently, serial twofold dilutions ofthe compounds in MHB were added to wells containing adherent organisms.The polystyrene plates were incubated for 20 h at 37° C. The MIC wastaken as the lowest drug concentration at which observable growth wasinhibited. To determine the MBC, the MHB containing antibiotics wasremoved from each well and replaced with antibiotic-free MHB; the plateswere incubated again for 20 h at 37° C. in air. The MBC was taken as thelowest concentration of each drug that resulted in no bacterial growthfollowing removal of the compounds.

TABLE 8 Antimicrobial activities of the compounds against Staphylococcusaureus ATCC 6538 planktonic and adherent cells Activity against Adherentorganisms Planktonic organisms (biofilm) (not in biofilm) MIC^(b)Compound MIC (μM) MBC (μM) (μM) MBC^(b) (μM) 2f 16 16 64 128 2j 64 64128 128 2d 128 128 128 >128 2i 128 128 128 128 2e 64 64 64 128Vancomycin NA NA 2 8 Linezolid NA NA 4 16

It is claimed:
 1. An inhibitor of Class II3-hydroxy-3-methylglutaryl-coenzyme A reductase having one of thefollowing structures:

wherein: R¹=is a hydroxyl, alkoxy, or substituted alkoxy; R²=is H,alkyl, or substituted alkyl, R⁴ and R⁶ are independently selected fromalkyl substituents of the type —(CH₂)_(m)—R^(C), wherein m=0-14,optionally having polar substituents such as OH, NR^(A)R^(B), OPO₃H₂,OSO₃H, PO₃H₂, SO₂H, and CO₂, optionally having unsaturation and/orbranching in the alkyl chain R⁵=OH, NR^(A)R^(B), or a halogen; R^(A) andR^(B) are independently selected from —(CH₂)_(n)H, —(CH₂)_(n)OH,—(CH₂)_(n)CH(CH₃)₂, —(CH₂)—C(CH₃)₃,—(CH₂)_(n)-cyclohexyl-(CH₂)_(n)-phenyl, wherein n=0-3, R^(C)=H, OH,NR^(A)R^(B), OPO₃H₂, OSO₂H, PO₃H₂, SO₂H, CO₂, CH(CH₃)₂, C(CH₃)₃,cyclohexyl, or phenyl X, Y, and Z are independently selected from N, CH,and CR⁵, such that the core ring structure is selected from benzene,pyridine, pyrazine, pyridazine, and pyrimidine; A, B, D, and E areindependently selected from N, CH, CR⁴, such that the core ringstructure is selected from benzene, pyridine, pyrazine, pyridazine, andpyrimidine; Q=S, O, or NR^(A), and U, V, and W are independentlyselected from N and CR⁵, such that the core ring structure is selectedfrom pyrrole, imidazole, pyrazole, furan, oxazole, isooxazole,thiophene, thiazole, and isothiazole, or a pharmaceutically acceptablesalts thereof, with the proviso that the inhibitor is not5-(N-(4-butylphenyl)sulfamoyl)-2-hydroxybenzoic acid.
 2. The inhibitorof claim 1, wherein R⁴ or R⁶ is a linear or branched alkyl having fromto 14 carbon atoms.
 3. The inhibitor of claim 1, wherein R¹ is OH. 4.The inhibitor of claim 1, wherein R¹ is —O—(CO)NH₂ or —OCH₂(CO)NH₂. 5.The inhibitor of claim 1, wherein R¹ is —OCH₂(CO)NH₂.
 6. The inhibitorof claim 1, wherein A, B, D and E are part of a benzene ring structure.7. The inhibitor of claim 1 selected from 2f, 2l, 2j, and 2e, andpharmaceutically acceptable salts thereof.
 8. An inhibitor according toclaim 1, the inhibitor having antimicrobial activity against one or morebacterial or fungal pathogens.
 9. The inhibitor of claim 1, wherein thebacterial pathogen is a Gram-positive pathogen.
 10. The inhibitor ofclaim 8, wherein the pathogen is selected from the group consisting ofBacillus anthracis, Bacillus subtilis, Bacillus cereus, Listeriamonocytogenes, Brucella abortus, Candida albicans, Staphylococcusaureus, Enterococcus faecalis and Streptococcus pneumoniae.
 11. Theinhibitor according to claim 8, wherein the pathogen is resistant tovancomycin and/or methicillin.
 12. An inhibitor according to claim 1,wherein the inhibitor exhibits no toxicity or low toxicity towardmammalian cells.
 13. An pharmaceutical composition comprising: apharmaceutically acceptable excipient; and a compound according to claim1 or 5-(N-(4-butylphenyl)sulfamoyl)-2-hydroxybenzoic acid.
 14. A methodof treating a multicellular organism infected with a bacterial or fungalpathogen comprising administering to the organism the pharmaceuticalcomposition of claim 8 in an amount effective to kill or inhibit growthof the pathogen.
 15. The method of claim 14, wherein the organism is ahuman.
 16. A biocide comprising: a diluent; and a compound according toclaim 1 or 5-(N-(4-butylphenyl)sulfamoyl)-2-hydroxybenzoic acid.
 17. Amethod of disinfecting a surface comprising or at risk for comprisingplanktonic or adherent pathogenic bacteria comprising contacting thesurface with an effective amount of the biocide of claim 16 for a periodof time sufficient to kill or inhibit growth of the bacteria.